Receptor that binds trail

ABSTRACT

A protein designated TRAIL receptor binds the protein known as TNF-Related Apoptosis-Inducing Ligand (TRAIL). The TRAIL receptor finds use in purifying TRAIL or inhibiting activities thereof. Isolated DNA sequences encoding TRAIL-R polypeptides are provided, along with expression vectors containing the DNA sequences, and host cells transformed with such recombinant expression vectors. Antibodies that are immunoreactive with TRAIL-R are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/869,852 filed Jun. 4, 1997 now abandoned, which is acontinuation-in-part of application Ser. No. 08/829,536, filed Mar. 28,1997, now abandoned, which is a continuation-in-part of application Ser.No. 08/815,255, filed Mar. 12, 1997, now abandoned, which is acontinuation-in-part of application Ser. No. 08/799,861, filed Feb. 13,1997, now abandoned.

BACKGROUND OF THE INVENTION

A protein known as TNF-related apoptosis-inducing ligand (TRAIL) is amember of the tumor necrosis factor family of ligands (Wiley et al.,Immunity, 3:673-682, 1995). TRAIL has demonstrated the ability to induceapoptosis of certain transformed cells, including a number of differenttypes of cancer cells as well as virally infected cells (PCT applicationWO 97/01633 and Wiley et al., supra).

Identification of receptor protein(s) that bind TRAIL would prove usefulin further study of the biological activities of TRAIL. However, priorto the present invention, no receptor for TRAIL had been reported.

SUMMARY OF THE INVENTION

The present invention is directed to a novel protein designated TRAILreceptor (TRAIL-R), which binds to a protein known as TNF-relatedapoptosis-inducing ligand (TRAIL). DNA encoding TRAIL-R, and expressionvectors comprising such DNA, are provided. A method for producingTRAIL-R polypeptides comprises culturing host cells transformed with arecombinant expression vector encoding TRAIL-R, under conditions thatpromote expression of TRAIL-R, then recovering the expressed TRAIL-Rpolypeptides from the culture. Antibodies that are immunoreactive withTRAIL-R are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the nucleotide sequence of a human TRAIL receptor DNAfragment SEQ ID NO:3, as well as the amino acid sequence encoded therebySEQ ID NO:4. This DNA fragment is described in Example 3.

FIG. 2 presents the results of the assay described in example 7. In theassay, a soluble TRAIL-R/Fc fusion protein blocked TRAIL-inducedapoptosis of Jurkat cells.

FIG. 3 presents the results of the experiment described in example 8.The indicated compounds were demonstrated to inhibit apoptosis of cellsexpressing TRAIL receptor.

DETAILED DESCRIPTION OF THE INVENTION

A novel protein designated TRAIL receptor (TRAIL-R) is provided herein.TRAIL-R binds to the cytokine designated TNF-related apoptosis-inducingligand (TRAIL). Certain uses of TRAIL-R flow from this ability to bindTRAIL, as discussed further below. TRAIL-R finds use in inhibitingbiological activities of TRAIL, or in purifying TRAIL by affinitychromatography, for example.

The nucleotide sequence of the coding region of a human TRAIL receptorDNA is presented in SEQ ID NO: 1. The amino acid sequence encoded by theDNA sequence of SEQ ID NO:1 is shown in SEQ ID NO:2. This sequenceinformation identifies the TRAIL receptor protein as a member of thetumor necrosis factor receptor (TNF-R) family of receptors (reviewed inSmith et al., Cell 76:959-962, 1994) The extracellular domain containscysteine rich repeats; such motifs have been reported to be importantfor ligand binding in other receptors of this family. TRAIL-R contains aso-called "death domain" in the cytoplasmic region; such domains incertain other receptors are associated with transduction of apoptoticsignals. These and other features of the protein are discussed in moredetail below.

TRAIL-R protein or immunogenic fragments thereof may be employed asimmunogens to generate antibodies that are immunoreactive therewith. Inone embodiment of the invention, the antibodies are monoclonalantibodies.

A human TRAIL-R protein was purified as described in example 1. Inexample 2, amino acid sequence information derived from fragments ofTRAIL-R is presented. One embodiment of the invention is directed to apurified human TRAIL-R protein that is capable of binding TRAIL, whereinthe TRAIL-R is characterized as comprising the amino acid sequenceVPANEGD (amino acids 327 to 333 of SEQ ID NO:2). In another embodiment,the TRAIL-R additionally comprises the sequenceETLRQCFDDFADLVPFDSWEPLMRKLGLMDNEIKVAKAEAAGHRDTLXTML (amino acids 336 to386 of SEQ ID NO:2, with one unknown amino acid indicated as X). Alsoprovided are TRAIL-R fragments comprising only one of thesecharacterizing amino acid sequences.

The nucleotide sequence of a TRAIL-R DNA fragment, and the amino acidsequence encoded thereby, are presented in FIG. 1 (SEQ ID NO:3 and SEQID NO:4); see example 3. The amino acid sequence presented in FIG. 1 hascharacteristics of the so-called "death domains" found in thecytoplasmic region of certain other receptor proteins. Such domains havebeen reported to be associated with transduction of apoptotic signals.Cytoplasmic death domains have been identified in Fas antigen (Itoh andNagata, J. Biol. Chem. 268:10932, 1993), TNF receptor type I (Tartagliaet al. Cell 74:845, 1993), DR3 (Chinnaiyan et al., Science 274:990-992,1996), and CAR-1 (Brojatsch et al., Cell 87:845-855, 1996). The role ofthese death domains in initiating intracellular apoptotic signalingcascades is discussed further below.

SEQ ID NO:1 presents the nucleotide sequence of the coding region of ahuman TRAIL receptor DNA, including an initiation codon (ATG) and atermination codon (TAA). The amino acid sequence encoded by the DNA ofSEQ ID NO:1 is presented in SEQ ID NO:2. The fragment depicted in FIG. 1corresponds to the region of TRAIL-R that is presented as amino acids336 to 386 in SEQ ID NO:2.

The TRAIL-R protein of SEQ ID NO:2 includes an N-terminal hydrophobicregion that functions as a signal peptide, followed by an extracellulardomain, a transmembrane region comprising amino acids 211 through 23 1,and a C-terminal cytoplasmic domain. Computer analysis predicts that thesignal peptide corresponds to residues 1 to 51 of SEQ ID NO:2. Cleavageof the signal peptide thus would yield a mature protein comprising aminoacids 52 through 440 of SEQ ID NO:2. The calculated molecular weight fora mature protein containing residues 52 to 440 of SEQ ID NO:2 is about43 kilodaltons. The next most likely computer-predicted signal peptidasecleavage sites (in descending order) occur after amino acids 50 and 58of SEQ ID NO:2.

In another embodiment of the invention, the N-terminal residue of amature TRAIL-R protein is the isoleucine residue at position 56 of SEQID NO:2. Sequences of several tryptic digest peptide fragments ofTRAIL-R were determined by a combination of N-terminal sequencing andNano-ES MS/MS (nano electrospray tandem mass spectrometry). TheN-terminal amino acid of one of the peptide fragments was the isoleucineat position 56 of SEQ ID NO:2. Since this fragment was not preceded by atrypsin cleavage site, the (Ile)56 residue may correspond to theN-terminal residue resulting from cleavage of the signal peptide.

A further embodiment of the invention is directed to mature TRAIL-Rhaving amino acid 54 as the N-terminal residue. In one preparation ofTRAIL-R (a soluble TRAIL-R/Fc fusion protein expressed in CV1-EBNAcells), the signal peptide was cleaved after residue 53 of SEQ ID NO:2.

The skilled artisan will recognize that the molecular weight ofparticular preparations of TRAIL-R protein may differ, according to suchfactors as the degree of glycosylation. The glycosylation pattern of aparticular preparation of TRAIL-R may vary according to the type ofcells in which the protein is expressed, for example. Further, a givenpreparation may include multiple differentially glycosylated species ofthe protein. TRAIL-R polypeptides with or without associatednative-pattern glycosylation are provided herein. Expression of TRAIL-Rpolypeptides in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules.

In one embodiment, the protein is characterized by a molecular weightwithin the range of about 50 to 55 kilodaltons, which is the molecularweight determined for a preparation of native, full length, humanTRAIL-R. Molecular weight can be determined by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE).

Example 1 presents one method for purifying a TRAIL-R protein. Jurkatcells are disrupted, and the subsequent purification process includesaffinity chromatography (employing a chromatography matrix containingTRAIL), and reversed phase HPLC.

TRAIL-R polypeptides of the present invention may be purified by anysuitable alternative procedure, using known protein purificationtechniques. In one alternative procedure, the chromatography matrixinstead comprises an antibody that binds TRAIL-R. Other cell typesexpressing TRAIL-R (e.g., the PS-1 cells described in example 2) can besubstituted for the Jurkat cells. The cells can be disrupted by any ofthe numerous known techniques, including freeze-thaw cycling,sonication, mechanical disruption, or by use of cell lysing agents.

The desired degree of purity depends on the intended use of the protein.A relatively high degree of purity is desired when the protein is to beadministered ill vivo, for example. Advantageously, TRAIL-R polypeptidesare purified such that no protein bands corresponding to other(non-TRAIL-R) proteins are detectable upon analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognizedby one skilled in the pertinent field that multiple bands correspondingto TRAIL-R protein may be visualized by SDS-PAGE, due to differentialglycosylation, differential post-translational processing, and the like.TRAIL-R most preferably is purified to substantial homogeneity, asindicated by a single protein band upon analysis by SDS-PAGE. Theprotein band may be visualized by silver staining, Coomassie bluestaining, or (if the protein is radiolabeled) by autoradiography.

The present invention encompasses TRAIL-R in various forms, includingthose that are naturally occurring or produced through varioustechniques such as procedures involving recombinant DNA technology. Suchforms of TRAIL-R include, but are not limited to, fragments,derivatives, variants, and oligomers of TRAIL-R, as well as fusionproteins containing TRAIL-R or fragments thereof.

TRAIL-R may be modified to create derivatives thereof by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of TRAIL-R may be prepared by linking the chemical moietiesto functional groups on TRAIL-R amino acid side chains or at theN-terminus or C-terminus of a TRAIL-R polypeptide. Conjugates comprisingdiagnostic (detectable) or therapeutic agents attached to TRAIL-R arecontemplated herein, as discussed in more detail below.

Other derivatives of TRAIL-R within the scope of this invention includecovalent or aggregative conjugates of TRAIL-R polypeptides with otherproteins or polypeptides, such as by synthesis in recombinant culture asN-terminal or C-terminal fusions. Examples of fusion proteins arediscussed below in connection with TRAIL-R oligomers. Further,TRAIL-R-containing fusion proteins can comprise peptides added tofacilitate purification and identification of TRAIL-R. Such peptidesinclude, for example, poly-His or the antigenic identification peptidesdescribed in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology6:1204, 1988. One such peptide is the Flag® peptide,Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, which is highly antigenic and providesan epitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinant protein. Amurine hybridoma designated 4E11 produces a monoclonal antibody thatbinds the Flag® peptide in the presence of certain divalent metalcations, as described in U.S. Pat. No. 5,011,912, hereby incorporated byreference. The 4E11 hybridoma cell line has been deposited with theAmerican Type Culture Collection under accession no. HB 9259. Monoclonalantibodies that bind the Flag® peptide are available from Eastman KodakCo., Scientific Imaging Systems Division, New Haven, Conn.

Both cell membrane-bound and soluble (secreted) forms of TRAIL-R areprovided herein. Soluble TRAIL-R may be identified (and distinguishedfrom non-soluble membrane-bound counterparts) by separating intact cellsexpressing a TRAIL-R polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired protein. The presence of TRAIL-R in the medium indicatesthat the protein was secreted from the cells and thus is a soluble formof the desired protein.

Soluble forms of receptor proteins typically lack the transmembraneregion that would cause retention of the protein on the cell surface. Inone embodiment of the invention, a soluble TRAIL-R polypeptide comprisesthe extracellular domain of the protein. A soluble TRAIL-R polypeptidemay include the cytoplasmic domain, or a portion thereof, as long as thepolypeptide is secreted from the cell in which it is produced. Oneexample of a soluble TRAIL-R is a soluble human TRAIL-R comprising aminoacids 52 to 210 of SEQ ID NO:2. Other soluble TRAIL-R polypeptidesinclude, but are not limited to, polypeptides comprising amino acids xto 210 of SEQ ID NO:2, wherein x is an integer from 51 through 59.

Soluble forms of TRAIL-R possess certain advantages over themembrane-bound form of the protein. Purification of the protein fromrecombinant host cells is facilitated, since the soluble proteins aresecreted from the cells. Further, soluble proteins are generally moresuitable for certain applications, e.g., for intravenous administration.

TRAIL-R fragments are provided herein. Such fragments may be prepared byany of a number of conventional techniques. Desired peptide fragmentsmay be chemically synthesized. An alternative involves generatingTRAIL-R fragments by enzymatic digestion, e.g., by treating the proteinwith an enzyme known to cleave proteins at sites defined by particularamino acid residues. Yet another suitable technique involves isolatingand amplifying a DNA fragment encoding a desired polypeptide fragment,by polymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed as the 5' and 3'primers in the PCR.

Examples of fragments are those comprising at least 20, or at least 30,contiguous amino acids of the sequence of SEQ ID NO:2. Fragments derivedfrom the cytoplasmic domain find use in studies of TRAIL-R-mediatedsignal transduction, and in regulating cellular processes associatedwith transduction of biological signals. TRAIL-R polypeptide fragmentsalso may be employed as immunogens, in generating antibodies. Particularembodiments are directed to TRAIL-R polypeptide fragments that retainthe ability to bind TRAIL. Such a fragment may be a soluble TRAIL-Rpolypeptide, as described above.

Naturally occurring variants of the TRAIL-R protein of SEQ ID NO:2 areprovided herein. Such variants include, for example, proteins thatresult from alternate mRNA splicing events or from proteolytic cleavageof the TRAIL-R protein. Alternate splicing of mRNA may, for example,yield a truncated but biologically active TRAIL-R protein, such as anaturally occurring soluble form of the protein. Variations attributableto proteolysis include, for example, differences in the N- or C-terminiupon expression in different types of host cells, due to proteolyticremoval of one or more terminal amino acids from the TRAIL-R protein(generally from 1-5 terminal amino acids). TRAIL-R proteins in whichdifferences in amino acid sequence are attributable to geneticpolymorphism (allelic variation among individuals producing the protein)are also contemplated herein.

The skilled artisan will also recognize that the position(s) at whichthe signal peptide is cleaved may differ from that predicted by computerprogram, and may vary according to such factors as the type of hostcells employed in expressing a recombinant TRAIL-R polypeptide. Aprotein preparation may include a mixture of protein molecules havingdifferent N-terminal amino acids, resulting from cleavage of the signalpeptide at more than one site. As discussed above, particularembodiments of mature TRAIL-R proteins provided herein include, but arenot limited to, proteins having the residue at position 51, 52, 54, 56,or 59 of SEQ ID NO:2 as the N-terminal amino acid.

Regarding the discussion herein of various domains of TRAIL-R protein,the skilled artisan will recognize that the above-described boundariesof such regions of the protein are approximate. To illustrate, theboundaries of the transmembrane region (which may be predicted by usingcomputer programs available for that purpose) may differ from thosedescribed above. Thus, soluble TRAIL-R polypeptides in which theC-terminus of the extracellular domain differs from the residue soidentified above are contemplated herein.

Other naturally occurring TRAIL-R DNAs and polypeptides include thosederived from non-human species. Homologs of the human TRAIL-R of SEQ IDNO:2, from other mammalian species, are contemplated herein, forexample. Probes based on the human DNA sequence of SEQ ID NO:3 or SEQ IDNO:1 may be used to screen cDNA libraries derived from other mammalianspecies, using conventional cross-species hybridization techniques.

TRAIL-R DNA sequences may vary from the native sequences disclosedherein. Due to the known degeneracy of the genetic code, wherein morethan one codon can encode the same amino acid, a DNA sequence can varyfrom that shown in SEQ ID NO:1 and still encode a TRAIL-R protein havingthe amino acid sequence of SEQ ID NO:2. Such variant DNA sequences mayresult from silent mutations (e.g., occurring during PCR amplification),or may be the product of deliberate mutagenesis of a native sequence.Thus, among the DNA sequences provided herein are native TRAIL-Rsequences (e.g., cDNA comprising the nucleotide sequence presented inSEQ ID NO: 1) and DNA that is degenerate as a result of the genetic codeto a native TRAIL-R DNA sequence.

Among the TRAIL-R polypeptides provided herein are variants of nativeTRAIL-R polypeptides that retain a biological activity of a nativeTRAIL-R. Such variants include polypeptides that are substantiallyhomologous to native TRAIL-R, but which have an amino acid sequencedifferent from that of a native TRAIL-R because of one or moredeletions, insertions or substitutions. Particular embodiments include,but are not limited to, TRAIL-R polypeptides that comprise from one toten deletions, insertions or substitutions of amino acid residues, whencompared to a native TRAIL-R sequence. The TRAIL-R-encoding DNAs of thepresent invention include variants that differ from a native TRAIL-R DNAsequence because of one or more deletions, insertions or substitutions,but that encode a biologically active TRAIL-R polypeptide. Onebiological activity of TRAIL-R is the ability to bind TRAIL.

Nucleic acid molecules capable of hybridizing to the DNA of SEQ ID NO:1or SEQ ID NO:3 under moderately stringent or highly stringentconditions, and which encode a biologically active TRAIL-R, are providedherein. Such hybridizing nucleic acids include, but are not limited to,variant DNA sequences and DNA derived from non-human species, e.g.,non-human mammals.

Moderately stringent conditions include conditions described in, forexample Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed.,Vol. 1, pp 1.101-104, Cold Spring Harbor Laboratory Press, 1989.Conditions of moderate stringency, as defined by Sambrook et al.,include use of a prewashing solution of 5× SSC, 0.5% SDS, 1.0 mM EDTA(pH 8.0) and hybridization conditions of about 55° C., 5× SSC,overnight. Highly stringent conditions include higher temperatures ofhybridization and washing. One embodiment of the invention is directedto DNA sequences that will hybridize to the DNA of SEQ ID NOS:1 or 3under highly stringent conditions, wherein said conditions includehybridization at 68° C. followed by washing in 0.1× SSC/0.1% SDS at63-68° C.

Certain DNAs and polypeptides provided herein comprise nucleotide oramino acid sequences, respectively, that are at least 80% identical to anative TRAIL-R sequence. Also contemplated are embodiments in which aTRAIL-R DNA or polypeptide comprises a sequence that is at least 90%identical, at least 95% identical, or at least 98% identical to a nativeTRAIL-R sequence. The percent identity may be determined, for example,by comparing sequence information using the GAP computer program,version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984)and available from the University of Wisconsin Genetics Computer Group(UWGCG). The preferred default parameters for the GAP program include:(1) a unary comparison matrix (containing a value of 1 for identitiesand 0 for non-identities) for nucleotides, and the weighted comparisonmatrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, asdescribed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence andStructure, National Biomedical Research Foundation, pp. 353-358, 1979;(2) a penalty of 3.0 for each gap and an additional 0.10 penalty foreach symbol in each gap; and (3) no penalty for end gaps.

In particular embodiments of the invention, a variant TRAIL-Rpolypeptide differs in amino acid sequence from a native TRAIL-R, but issubstantially equivalent to a native TRAIL-R in a biological activity.One example is a variant TRAIL-R that binds TRAIL with essentially thesame binding affinity as does a native TRAIL-R. Binding affinity can bemeasured by conventional procedures, e.g., as described in U.S. Pat. No.5,512,457.

Variant amino acid sequences may comprise conservative substitution(s),meaning that one or more amino acid residues of a native TRAIL-R isreplaced by a different residue, but that the conservatively substitutedTRAIL-R polypeptide retains a desired biological activity of the nativeprotein (e.g., the ability to bind TRAIL). A given amino acid may bereplaced by a residue having similar physiochemical characteristics.Examples of conservative substitutions include substitution of onealiphatic residue for another, such as Ile, Val, Leu, or Ala for oneanother, or substitutions of one polar residue for another, such asbetween Lys and Arg; Glu and Asp; or Gln and Asn. Other conservativesubstitutions, e.g., involving substitutions of entire regions havingsimilar hydrophobicity characteristics, are well known.

In another example of variants, sequences encoding Cys residues that arenot essential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Certain receptors of the TNF-R family containcysteine-rich repeat motifs in their extracellular domains (Marsters etal., J. Biol. Chem. 267:5747-5750, 1992). These repeats are believed tobe important for ligand binding. To illustrate, Marsters et al., supra,reported that soluble TNF-R type I polypeptides lacking one of therepeats exhibited a ten fold reduction in binding affinity for TNFα andTNFβ; deletion of the second repeat resulted in a complete loss ofdetectable binding of the ligands. The human TRAIL-R of SEQ ID NO:2contains two such cysteine rich repeats, the first including residues 94through 137, and the second including residues 138 through 178. Cysteineresidues within these cysteine rich domains advantageously remainunaltered in TRAIL-R variants, when retention of TRAIL-binding activityis desired.

Other variants are prepared by modification of adjacent dibasic aminoacid residues, to enhance expression in yeast systems in which KEX2protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Mature human TRAIL-R contains such adjacent basic residuepairs at amino acids 72-73, 154-155, 322-323, 323-324, and 359-360 ofSEQ ID NO:2. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

TRAIL-R polypeptides, including variants and fragments thereof, can betested for biological activity in any suitable assay. The ability of aTRAIL-R polypeptide to bind TRAIL can be confirmed in conventionalbinding assays, examples of which are described below.

Expression Systems

The present invention also provides recombinant cloning and expressionvectors containing TRAIL-R DNA, as well as host cell containing therecombinant vectors. Expression vectors comprising TRAIL-R DNA may beused to prepare TRAIL-R polypeptides encoded by the DNA. A method forproducing TRAIL-R polypeptides comprises culturing host cellstransformed with a recombinant expression vector encoding TRAIL-R, underconditions that promote expression of TRAIL-R, then recovering theexpressed TRAIL-R polypeptides from the culture. The skilled artisanwill recognize that the procedure for purifying the expressed TRAIL-Rwill vary according to such factors as the type of host cells employed,and whether the TRAIL-R is membrane-bound or a soluble form that issecreted from the host cell.

Any suitable expression system may be employed. The vectors include aDNA encoding a TRAIL-R polypeptide, operably linked to suitabletranscriptional or translational regulatory nucleotide sequences, suchas those derived from a mammalian, microbial, viral, or insect gene.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, an mRNA ribosomal binding site, and appropriatesequences which control transcription and translation initiation andtermination. Nucleotide sequences are operably linked when theregulatory sequence functionally relates to the TRAIL-R DNA sequence.Thus, a promoter nucleotide sequence is operably linked to an TRAIL-RDNA sequence if the promoter nucleotide sequence controls thetranscription of the TRAIL-R DNA sequence. An origin of replication thatconfers the ability to replicate in the desired host cells, and aselection gene by which transformants are identified, are generallyincorporated into the expression vector.

In addition, a sequence encoding an appropriate signal peptide (nativeor heterologous) can be incorporated into expression vectors. A DNAsequence for a signal peptide (secretory leader) may be fused in frameto the TRAIL-R sequence so that the TRAIL-R is initially translated as afusion protein comprising the signal peptide. A signal peptide that isfunctional in the intended host cells promotes extracellular secretionof the TRAIL-R polypeptide. The signal peptide is cleaved from theTRAIL-R polypeptide upon secretion of TRAIL-R from the cell.

Suitable host cells for expression of TRAIL-R polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cellsare generally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce TRAIL-Rpolypeptides using RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a TRAIL-R polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant TRAIL-R polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. An appropriate promoter and a TRAIL-R DNA sequenceare inserted into the pBR322 vector. Other commercially availablevectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,Uppsala, Sweden) and pGEM1 (Promega Biotec. Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage k PL promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) andpPLc28 (resident in E. coli RR1, ATCC 53082).

TRAIL-R alternatively may be expressed in yeast host cells, preferablyfrom the Saccharomyces genus (e.g., S. cerevisiae). Other genera ofyeast, such as Pichia or Kluyveromyces, may also be employed. Yeastvectors will often contain an origin of replication sequence from a 2μyeast plasmid, an autonomously replicating sequence (ARS), a promoterregion, sequences for polyadenylation, sequences for transcriptiontermination, and a selectable marker gene. Suitable promoter sequencesfor yeast vectors include, among others, promoters for metallothionein,3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073,1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657. Another alternative is the glucose-repressible ADH2 promoterdescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast andE. coli may be constructed by insetting DNA sequences from pBR322 forselection and replication in E. coli (Amp^(r) gene and origin ofreplication) into the above-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the TRAIL polypeptide. The α-factor leader sequence is often insertedbetween the promoter sequence and the structural gene sequence. See,e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3' end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promotersequence may be grown for inducing expression in a "rich" medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the AH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems also may be employed toexpress recombinant TRAIL-R polypeptides. Bacculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

Expression vectors for use in mammalian host cells can be constructed asdisclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983), forexample. A useful system for stable high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A high expression vector, PMLSV N1/N4, described by Cosman etal., Nature 312:768, 1984 has been deposited as ATCC 39890. Additionalmammalian expression vectors are described in EP-A-0367566, and in WO91/18982. As one alternative, the vector may be derived from aretrovirus.

Overexpression of full length TRAIL-R has resulted in membrane blebbingand nuclear condensation of transfected CV-1/EBNA cells, indicating thatthe mechanism of cell death was apoptosis. For host cells in which suchTRAIL-R-mediated apoptosis occurs, a suitable apoptosis inhibitor may beincluded in the expression system.

To inhibit TRAIL-R-induced apoptosis of host cells expressingrecombinant TRAIL-R, the cells may be co-transfected with an expressionvector encoding a polypeptide that functions as an apoptosis inhibitor.Expression vectors encoding such polypeptides can be prepared byconventional procedures. Another approach involves adding an apoptosisinhibitor to the culture medium. The use of poxvirus CrmA, baculovirusP35, a C-terminal fragment of FADD, and the tripeptide derivativezVAD-fmk, to reduce death of host cells is illustrated in examples 6 and8.

zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) is atripeptide based compound, available from Enzyme System Products,Dublin, Calif. As illustrated in example 8, zVAD-fmk may be added to themedium in which cells expressing TRAIL-R are cultured.

The 38-kilodalton cowpox-derived protein that was subsequentlydesignated CrmA is described in Pickup et al. (Proc. Natl. Acad. Sci.USA 83:7698-7702, 1986; hereby incorporated by reference). Sequenceinformation for CrmA is presented in FIG. 4 of Pickup et al., supra. Oneapproach to producing and purifying CrmA protein is described in Ray etal. (Cell, 69:597-604, 1992; hereby incorporated by reference).

A 35-kilodalton protein encoded by Autographa californica nuclearpolyhedrosis virus, a baculovirus, is described in Friesen and Miller(J. Virol. 61:2264-2272, 1987; hereby incorporated by reference).Sequence information for this protein, designated baculovirus p35herein, is presented in FIG. 5 of Friesen and Miller, supra.

The death domain-containing cytoplasmic protein FADD (also known asMORT1) is described in Boldin et al. (J. Biol. Chem. 270:7795-7798,1995; hereby incorporated by reference). FADD has been reported toassociate, directly or indirectly, with the cytoplasmic death domain ofcertain receptors that mediate apoptosis (Boldin et al., Cell85:803-815, June 1996; Hsu et al., Cell 84:299-308, January 1996).

In one embodiment of the present invention, truncated FADD polypeptidesthat include the death domain (located in the C-terminal portion of theprotein), but lack the N-terminal region to which apoptosis effectorfunctions have been attributed, are employed to reduce apoptosis. Theuse of certain FADD deletion mutant polypeptides, truncated at theN-terminus, to inhibit death of cells expressing otherapoptosis-inducing receptors, is described in Hsu et al. (Cell84:299-308, 1996; hereby incorporated by reference).

This approach is illustrated in example 8, which employs one suitableFADD-dominant negative (FADD-DN) polypeptide, having an amino acidsequence corresponding to amino acids 117 through 245 of the MORT1 aminoacid sequence presented in Boldin et al. (J. Biol. Chem. 270:7795-7798,1995). In example 8, cells were co-transfected with a TRAIL-R-encodingexpression vector, and with an expression vector encoding theabove-described Flag® peptide, fused to the N-terminus of the FADD-DNpolypeptide.

While not wishing to be bound by theory, one possible explanation isthat the C-terminal fragments of FADD associate with the intracellulardeath domain of the receptor, but lack the N-terminal portion of theprotein that is necessary for effecting apoptosis (Hsu et al., Cell84:299-308, January 1996; Boldin et al., Cell 85:803-815, June 1996).The truncated FADD thereby may block association of endogenous, fulllength FADD with the receptor's death domain; consequently, theapoptosis that would be initiated by such endogenous FADD is inhibited.

Other apoptosis inhibitors useful in expression systems of the presentinvention can be identified in conventional assay procedures. One suchassay, in which compounds are tested for the ability to reduce apoptosisof cells expressing TRAIL-R, is described in example 8.

Poxvirus CrmA, baculovirus P35, and zVAD-fmk are viral caspascinhibitors. Other caspase inhibitors may be tested for the ability toreduce TRAIL-R-mediated cell death.

The use of CrmA, baculovirus p35, and certain peptide derivatives(including zVAD-fmk) as inhibitors of apoptosis in particularcells/systems is discussed in Sarin et al. (J. Exp. Med. 184:2445-2450,Dec. 1996; hereby incorporated by reference). The role of interleukin-1βconverting enzyme (ICE) family proteases in signal transduction cascadesleading to programmed cell death, and the use of inhibitors of suchproteases to block apoptosis, is discussed in Sarin et al., supra, andMuzio et al., Cell 85:817-827, 1996).

Apoptosis inhibitors generally need not be employed for expression ofTRAIL-R polypeptides lacking the cytoplasmic domain (i.e., lacking theregion of the protein involved in signal transduction). Thus, expressionsystems for producing soluble TRAIL-R polypeptides comprising only theextracellular domain (or a fragment thereof) need not include one of theabove-described apoptosis inhibitors.

Regarding signal peptides that may be employed in producing TRAIL-R, thenative signal peptide of TRAIL-R may be replaced by a heterologoussignal peptide or leader sequence, if desired. The choice of signalpeptide or leader may depend on factors such as the type of host cellsin which the recombinant TRAIL-R is to be produced. To illustrate,examples of heterologous signal peptides that are functional inmammalian host cells include the signal sequence for interleukin-7(IL-7) described in U.S. Pat. No. 4,965,195, the signal sequence forinterleukin-2 receptor described in Cosman et al., Nature 312:768(1984); the interleukin-4 receptor signal peptide described in EP367,566; the type I interleukin-1 receptor signal peptide described inU.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signalpeptide described in EP 460,846.

Oligomeric Forms of TRAIL-R

Encompassed by the present invention are oligomers that contain TRAIL-Rpolypeptides. TRAIL-R oligomers may be in the form of covalently-linkedor noncovalently-linked dimers, trimers, or higher oligomers.

One embodiment of the invention is directed to oligomers comprisingmultiple TRAIL-R polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the TRAIL-R polypeptides.Such peptides may be peptide linkers (spacers), or peptides that havethe property of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of TRAIL-R polypeptides attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourTRAIL-R polypeptides. The TRAIL-R moieties of the oligomer may besoluble polypeptides, as described above.

As one alternative, a TRAIL-R oligomer is prepared using polypeptidesderived from immunoglobulins. Preparation of fusion proteins comprisingcertain heterologous polypeptides fused to various portions ofantibody-derived polypeptides (including the Fe domain) has beendescribed, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn etal. (Nature 344:677, 1990); and Hollenbaugh and Aruffo ("Construction ofImmunoglobulin Fusion Proteins", in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11, 1992).

One embodiment of the present invention is directed to a TRAIL-R dimercomprising two fusion proteins created by fusing TRAIL-R to the Feregion of an antibody. A gene fusion encoding the TRAIL-R/Fc fusionprotein is inserted into an appropriate expression vector. TRAIL-R/Fcfusion proteins are expressed in host cells transformed with therecombinant expression vector, and allowed to assemble much likeantibody molecules, whereupon interchain disulfide bonds form betweenthe Fe moieties to yield divalent TRAIL-R.

Provided herein are fusion proteins comprising a TRAIL-R polypeptidefused to an Fe polypeptide derived from an antibody. DNA encoding suchfusion proteins, as well as dimers containing two fusion proteins joinedvia disulfide bonds between the Fe moieties thereof, are also provided.The term "Fe polypeptide" as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization are also included.

One suitable Fe polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fe region of a human IgG1 antibody. Another useful Fe polypeptide isthe Fe mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,(EMBO J. 13:3992-4001, 1994). The amino acid sequence of this mutein isidentical to that of the native Fe sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fe receptors.

In other embodiments, TRAIL-R may be substituted for the variableportion of an antibody heavy or light chain. If fusion proteins are madewith both heavy and light chains of an antibody, it is possible to forma TRAIL-R oligomer with as many as four TRAIL-R extracellular regions.

Alternatively, the oligomer is a fusion protein comprising multipleTRAIL-R polypeptides, with or without peptide linkers (spacer peptides).Among the suitable peptide linkers are those described in U.S. Pat. Nos.4,751,180 and 4,935,233, which are hereby incorporated by reference. ADNA sequence encoding a desired peptide linker may be inserted between,and in the same reading frame as, the DNA sequences encoding TRAIL-R,using any suitable conventional technique. For example, a chemicallysynthesized oligonucleotide encoding the linker may be ligated betweensequences encoding TRAIL-R. In particular embodiments, a fusion proteincomprises from two to four soluble TRAIL-R polypeptides, separated bypeptide linkers.

Another method for preparing oligomeric TRAIL-R involves use of aleucine zipper. Leucine zipper domains are peptides that promoteoligomerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, 1988), and have since been found in a varietyof different proteins. Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.

Examples of leucine zipper domains suitable for producing solubleoligomeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al. (FEBS Letters 344:191, 1994), herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al. (Semin. Immunol. 6:267-278, 1994).Recombinant fusion proteins comprising a soluble TRAIL-R polypeptidefused to a leucine zipper peptide are expressed in suitable host cells,and the soluble oligomeric TRAIL-R that forms is recovered from theculture supernatant.

Oligomeric TRAIL-R has the property of bivalent, trivalent, etc. bindingsites for TRAIL. The above-described fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns. DNA sequences encoding oligomeric TRAIL-R, or encoding fusionproteins useful in preparing TRAIL-R oligomers, are provided herein.

Assays

TRAIL-R proteins (including fragments, variants, oligomers, and otherforms of TRAIL-R) may be tested for the ability to bind TRAIL in anysuitable assay, such as a conventional binding assay. To illustrate,TRAIL-R may be labeled with a detectable reagent (e.g., a radionuclide,chromophore, enzyme that catalyzes a calorimetric or fluorometricreaction, and the like). The labeled TRAIL-R is contacted with cellsexpressing TRAIL. The cells then are washed to remove unbound labeledTRAIL-R, and the presence of cell-bound label is determined by asuitable technique, chosen according to the nature of the label.

One example of a binding assay procedure is as follows. A recombinantexpression vector containing TRAIL cDNA is constructed, e.g., asdescribed in in PCT application WO 97/01633, hereby incorporated byreference. DNA and amino acid sequence information for human and mouseTRAIL is presented in WO 97/01633. TRAIL comprises an N-terminalcytoplasmic domain, a transmembrane region, and a C-terminalextracellular domain. CV1-EBNA-1 cells in 10 cm² dishes are transfectedwith the recombinant expression vector. CV-1/EBNA-1 cells (ATCC CRL10478) constitutively express EBV nuclear antigen-i driven from the CMVimmediate-early enhancer/promoter. CV1-EBNA-1 was derived from theAfrican Green Monkey kidney cell line CV-1 (ATCC CCL 70), as describedby McMahan et al. (EMBO J. 10:2821, 1991).

The transfected cells are cultured for 24 hours, and the cells in eachdish then are split into a 24-well plate. After culturing an additional48 hours, the transfected cells (about 4×10⁴ cells/well) are washed withBM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovineserum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50mg/ml nonfat dry milk has been added. The cells then are incubated for 1hour at 37° C. with various concentrations of a soluble TRAIL-R/Fcfusion protein. Cells then are washed and incubated with a constantsaturating concentration of a ¹²⁵ I-mouse anti-human IgG in bindingmedium, with gentle agitation for 1 hour at 37° C. After extensivewashing, cells are released via trypsinization.

The mouse anti-human IgG employed above is directed against the Fcregion of human IgG and can be obtained from Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa. The antibody is radioiodinated usingthe standard chloramine-T method. The antibody will bind to the Fcportion of any TRAIL-R/Fc protein that has bound to the cells. In allassays, non-specific binding of ¹²⁵ I-antibody is assayed in the absenceof TRAIL-R/Fc, as well as in the presence of TRAIL-R/Fc and a 200-foldmolar excess of unlabeled mouse anti-human IgG antibody.

Cell-bound ¹²⁵ I-antibody is quantified on a Packard Autogamma counter.Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) aregenerated on RS/1 (BBN Software, Boston, Mass.) run on a Microvaxcomputer.

Another type of suitable binding assay is a competitive binding assay.To illustrate, biological activity of a TRAIL-R variant may bedetermined by assaying for the variant's ability to compete with anative TRAIL-R for binding to TRAIL.

Competitive binding assays can be performed by conventional methodology.Reagents that may be employed in competitive binding assays includeradiolabeled TRAIL-R and intact cells expressing TRAIL (endogenous orrecombinant) on the cell surface. For example, a radiolabeled solubleTRAIL-R fragment can be used to compete with a soluble TRAIL-R variantfor binding to cell surface TRAIL. Instead of intact cells, one couldsubstitute a soluble TRAIL/Fc fusion protein bound to a solid phasethrough the interaction of Protein A or Protein G (on the solid phase)with the Fe moiety. Chromatography columns that contain Protein A andProtein G include those available from Pharmacia Biotech, Inc.,Piscataway, N.J. Another type of competitive binding assay utilizesradiolabeled soluble TRAIL, such as a soluble TRAIL/FC fusion protein,and intact cells expressing TRAIL-R. Qualitative results can be obtainedby competitive autoradiographic plate binding assays, while Scatchardplots (Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) may be utilized togenerate quantitative results.

Another type of assay for biological activity involves testing a TRAIL-Rpolypeptide for the ability to block TRAIL-mediated apoptosis of targetcells, such as the human leukemic T-cell line known as Jurkat cells, forexample. TRAIL-mediated apoptosis of the cell line designated Jurkatclone E6-1 (ATCC TIB 152) is demonstrated in assay procedures describedin PCT application WO 97/01633, hereby incorporated by reference.

Uses of TRAIL-R

Uses of TRAIL-R include, but are not limited to, the following. Certainof these uses of TRAIL-R flow from its ability to bind TRAIL.

TRAIL-R finds use as a protein purification reagent. TRAIL-Rpolypeptides may be attached to a solid support material and used topurify TRAIL proteins by affinity chromatography. In particularembodiments, a TRAIL-R polypeptide (in any form described herein that iscapable of binding TRAIL) is attached to a solid support by conventionalprocedures. As one example, chromatography columns containing functionalgroups that will react with functional groups on amino acid side chainsof proteins are available (Pharmacia Biotech, Inc., Piscataway, N.J.).In an alternative, a TRAIL-R/Fc protein is attached to Protein A- orProtein G-containing chromatography columns through interaction with theFe moiety.

TRAIL-R proteins also find use in measuring the biological activity ofTRAIL proteins in terms of their binding affinity for TRAIL-R. TRAIL-Rproteins thus may be employed by those conducting "quality assurance"studies, e.g., to monitor shelf life and stability of TRAIL proteinunder different conditions. To illustrate, TRAIL-R may be employed in abinding affinity study to measure the biological activity of a TRAILprotein that has been stored at different temperatures, or produced indifferent cell types. TRAIL-R also may be used to determine whetherbiological activity is retained after modification of a TRAIL protein(e.g., chemical modification, truncation, mutation, etc.). The bindingaffinity of the modified TRAIL protein for TRAIL-R is compared to thatof an unmodified TRAIL protein to detect any adverse impact of themodifications on biological activity of TRAIL. The biological activityof a TRAIL protein thus can be ascertained before it is used in aresearch study, for example.

TRAIL-R also finds use in purifying or identifying cells that expressTRAIL on the cell surface. TRAIL-R polypeptides are bound to a solidphase such as a column chromatography matrix or a similar suitablesubstrate. For example, magnetic microspheres can be coated with TRAIL-Rand held in an incubation vessel through a magnetic field. Suspensionsof cell mixtures containing TRAIL-expressing cells are contacted withthe solid phase having TRAIL-R thereon. Cells expressing TRAIL on thecell surface bind to the fixed TRAIL-R, and unbound cells then arewashed away.

Alternatively, TRAIL-R can be conjugated to a detectable moiety, thenincubated with cells to be tested for TRAIL expression. Afterincubation, unbound labeled TRAIL-R is removed and the presence orabsence of the detectable moiety on the cells is determined.

In a further alternative, mixtures of cells suspected of containingTRAIL⁺ cells are incubated with biotinylated TRAIL-R. Incubation periodsare typically at least one hour in duration to ensure sufficientbinding. The resulting mixture then is passed through a column packedwith avidin-coated beads, whereby the high affinity of biotin for avidinprovides binding of the desired cells to the beads. Procedures for usingavidin-coated beads are known (see Berenson, et al. J. Cell. Biochem.,10D:239, 1986). Washing to remove unbound material, and the release ofthe bound cells, are performed using conventional methods.

TRAIL-R polypeptides also find use as carriers for delivering agentsattached thereto to cells bearing TRAIL. Cells expressing TRAIL includethose identified in Wiley et al. (Immunity, 3:673-682, 1995). TRAIL-Rproteins thus can be used to deliver diagnostic or therapeutic agents tosuch cells (or to other cell types found to express TRAIL on the cellsurface) in in vitro or in vivo procedures.

Detectable (diagnostic) and therapeutic agents that may be attached to aTRAIL-R polypeptide include, but are not limited to, toxins, othercytotoxic agents, drugs, radionuclides, chromophores, enzymes thatcatalyze a calorimetric or fluorometric reaction, and the like, with theparticular agent being chosen according to the intended application.Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins suchas trichothecenes, and derivatives and fragments (e.g., single chains)thereof. Radionuclides suitable for diagnostic use include, but are notlimited to, ¹²³ I, ¹³¹ I, ^(99m) Tc, ¹¹¹ In, and ⁷⁶ Br. Examples ofradionuclides suitable for therapeutic use arc ¹³¹ I, ²¹¹ At, ⁷⁷ Br, ⁸⁶Re, ¹⁸⁸ Re, ²¹² Pb, ²¹² Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, and ⁶⁷ Cu.

Such agents may be attached to the TRAIL-R by any suitable conventionalprocedure. TRAIL-R, being a protein, comprises functional groups onamino acid side chains that can be reacted with functional groups on adesired agent to form covalent bonds, for example. Alternatively, theprotein or agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for attachingvarious molecules to proteins (Pierce Chemical Company, Rockford, Ill.).A number of techniques for radiolabeling proteins are known.Radionuclide metals may be attached to TRAIL-R by using a suitablebifunctional chelating agent, for example.

Conjugates comprising TRAIL-R and a suitable diagnostic or therapeuticagent (preferably covalently linked) are thus prepared. The conjugatesare administered or otherwise employed in an amount appropriate for theparticular application.

TRAIL-R DNA and polypeptides of the present invention may be used indeveloping treatments for any disorder mediated (directly or indirectly)by defective, or insufficient amounts of, TRAIL-R. TRAIL-R polypeptidesmay be administered to a mammal afflicted with such a disorder.Alternatively, a gene therapy approach may be taken. Disclosure hereinof native TRAIL-R nucleotide sequences permits the detection ofdefective TRAIL-R genes, and the replacement thereof with normalTRAIL-R-encoding genes. Defective genes may be detected in in vitrodiagnostic assays, and by comparision of a native TRAIL-R nucleotidesequence disclosed herein with that of a TRAIL-R gene derived from aperson suspected of harboring a defect in this gene.

Another use of the protein of the present invention is as a researchtool for studying the biological effects that result from inhibitingTRAIL/TRAIL-R interactions on different cell types. TRAIL-R polypeptidesalso may be employed in in vitro assays for detecting TRAIL or TRAIL-Ror the interactions thereof.

TRAIL-R may be employed in inhibiting a biological activity of TRAIL, inin vitro or in vivo procedures. A purified TRAIL-R polypeptide may beused to inhibit binding of TRAIL to endogenous cell surface TRAIL-R.Biological effects that result from the binding of TRAIL to endogenousreceptors thus are inhibited. Various forms of TRAIL-R may be employed,including, for example, the above-described TRAIL-R fragments,oligomers, derivatives, and variants that are capable of binding TRAIL.In one embodiment, a soluble TRAIL-R is employed to inhibit a biologicalactivity of TRAIL, e.g., to inhibit TRAIL-mediated apoptosis ofparticular cells.

TRAIL-R may be administered to a mammal to treat a TRAIL-mediateddisorder. Such TRAIL-mediated disorders include conditions caused(directly or indirectly) or exacerbated by TRAIL.

TRAIL-R may be useful for treating thrombotic microangiopathies. Onesuch disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, H.C., Semin. Hematol., 24:71, 1987; Thompson et al., Blood, 80:1890,1992). Increasing TTP-associated mortality rates have been reported bythe U.S. Centers for Disease Control (Torok et al., Am. J. Hematol.50:84, 1995).

Plasma from patients afflicted with TTP (including HIV⁺ and HIV⁻patients) induces apoptosis of human endothelial cells of dermalmicrovascular origin, but not large vessel origin (Laurence et al.,Blood, 87:3245, Apr. 15, 1996). Plasma of TTP patients thus is thoughtto contain one or more factors that directly or indirectly induceapoptosis. As described in PCT application WO 97/01633 (herebyincorporated by reference), TRAIL is present in the serum of TTPpatients, and may play a role in inducing apoptosis of microvascularendothelial cells.

Another thrombotic microangiopathy is hemolytic-uremic syndrome (HUS)(Moake, J. L., Lancet, 343:393, 1994; Melnyk et al., (Arch. Intern.Med., 155:2077, 1995; Thompson et al., supra). One embodiment of theinvention is directed to use of TRAIL-R to treat the condition that isoften referred to as "adult HUS" (even though it can strike children aswell). A disorder known as childhood/diarrhea-associated HUS differs inetiology from adult HUS.

Other conditions characterized by clotting of small blood vessels may betreated using TRAIL-R. Such conditions include but are not limited tothe following. Cardiac problems seen in about 5-10% of pediatric AIDSpatients are believed to involve clotting of small blood vessels.Breakdown of the microvasculature in the heart has been reported inmultiple sclerosis patients. As a further example, treatment of systemiclupus erythematosus (SLE) is contemplated.

In one embodiment, a patient's blood or plasma is contacted with TRAIL-Rex vivo. The TRAIL-R may be bound to a suitable chromatography matrix byconventional procedures. The patient's blood or plasma flows through achromatography column containing TRAIL-R bound to the matrix, beforebeing returned to the patient. The immobilized receptor binds TRAIL,thus removing TRAIL protein from the patient's blood.

Alternatively, TRAIL-R may be administered in vivo to a patientafflicted with a thrombotic microangiopathy. In one embodiment, asoluble form of TRAIL-R is administered to the patient.

The present invention thus provides a method for treating a thromboticmicroangiopathy, involving use of an effective amount of TRAIL-R. ATRAIL-R polypeptide may be employed in in vivo or ex vivo procedures, toinhibit TRAIL-mediated damage to (e.g., apoptosis of) microvascularendothelial cells.

TRAIL-R may be employed in conjunction with other agents useful intreating a particular disorder. In an in vitro study reported byLaurence et al. (Blood 87:3245, 1996), some reduction of TTPplasma-mediated apoptosis of microvascular endothelial cells wasachieved by using an anti-Fas blocking antibody, aurintricarboxylicacid, or normal plasma depleted of cryoprecipitate.

Thus, a patient may be treated with an agent that inhibitsFas-ligand-mediated apoptosis of endothelial cells, in combination withan agent that inhibits TRAIL-mediated apoptosis of endothelial cells. Inone embodiment, TRAIL-R and an anti-FAS blocking antibody are bothadministered to a patient afflicted with a disorder characterized bythrombotic microangiopathy, such as TTP or HUS. Examples of blockingmonoclonal antibodies directed against Fas antigen (CD95) are describedin PCT application publication number WO 95/10540, hereby incorporatedby reference.

Another embodiment of the present invention is directed to the use ofTRAIL-R to reduce TRAIL-mediated death of T cells in HIV-infectedpatients. The role of T cell apoptosis in the development of AIDS hasbeen the subject of a number of studies (see, for example, Meyaard etal., Science 257:217-219, 1992; Groux et al., J Exp. Med., 175:331,1992; and Oyaizu et al., in Cell Activation and Apoptosis in HIVInfection, Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp.101-114). Certain investigators have studied the role of Fas-mediatedapoptosis; the involvement of interleukin-1β-converting enzyme (ICE)also has been explored (Estaquier et al., Blood 87:4959-4966, 1996;Mitra et al., Immunology 87:581-585, 1996; Katsikis et al., J. Exp. Med.181:2029-2036, 1995). It is possible that T cell apoptosis occursthrough multiple mechanisms.

At least some of the T cell death seen in HIV⁺ patients is believed tobe mediated by TRAIL. While not wishing to be bound by theory, suchTRAIL-mediated T cell death is believed to occur through the mechanismknown as activation-induced cell death (AICD).

Activated human T cells are induced to undergo programmed cell death(apoptosis) upon triggering through the CD3/T cell receptor complex, aprocess termed activated-induced cell death (AICD). AICD of CD4⁺ T cellsisolated from HIV-infected aymptomatic individuals has been reported(Groux et al., supra). Thus, AICD may play a role in the depletion ofCD4⁺ T cells and the progression to AIDS in HIV-infected individuals.

The present invention provides a method of inhibiting TRAIL-mediated Tcell death in HIV⁺ patients, comprising administering TRAIL-R(preferably, a soluble TRAIL-R polypeptide) to the patients. In oneembodiment, the patient is asymptomatic when treatment with TRAIL-Rcommences. If desired, prior to treatment, peripheral blood T cells maybe extracted from an HIV⁺ patient, and tested for susceptibility toTRAIL-mediated cell death by conventional procedures.

In one embodiment, a patient's blood or plasma is contacted with TRAIL-Rex vivo. The TRAIL-R may be bound to a suitable chromatography matrix byconventional procedures. The patient's blood or plasma flows through achromatography column containing TRAIL-R bound to the matrix, beforebeing returned to the patient. The immobilized TRAIL-R binds TRAIL, thusremoving TRAIL protein from the patient's blood.

In treating HIV⁺ patients, TRAIL-R may be employed in combination withother inhibitors of T cell apoptosis. Fas-mediated apoptosis also hasbeen implicated in loss of T cells in HIV⁺ individuals (Katsikis et al.,J. Exp. Med. 181:2029-2036, 1995). Thus, a patient susceptible to bothFas ligand (Fas-L)-mediated and TRAIL-mediated T cell death may betreated with both an agent that blocks TRAIL/TRAIL-R interactions and anagent that blocks Fas-L/Fas interactions. Suitable agents for blockingbinding of Fas-L to Fas include, but are not limited to, soluble Faspolypeptides; oligomeric forms of soluble Fas polypeptides (e.g., dimersof sFas/Fc); anti-Fas antibodies that bind Fas without transducing thebiological signal that results in apoptosis; anti-Fas-L antibodies thatblock binding of Fas-L to Fas; and muteins of Fas-L that bind Fas butdon't transduce the biological signal that results in apoptosis.Preferably, the antibodies employed in the method are monoclonalantibodies. Examples of suitable agents for blocking Fas-L/Fasinteractions, including blocking anti-Fas monoclonal antibodies, aredescribed in WO 95/10540, hereby incorporated by reference.

Compositions comprising an effective amount of a TRAIL-R polypeptide ofthe present invention, in combination with other components such as aphysiologically acceptable diluent, carrier, or excipient, are providedherein. TRAIL-R can be formulated according to known methods used toprepare pharmaceutically useful compositions. TRAIL-R can be combined inadmixture, either as the sole active material or with other known activematerials suitable for a given indication, with pharmaceuticallyacceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphatebuffered solutions), preservatives (e.g., thimerosal, benzyl alcohol,parabens), emulsifiers, solubilizers, adjuvants and/or carriers.Suitable formulations for pharmaceutical compositions include thosedescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Company, Easton, Pa.

In addition, such compositions can contain TRAIL-R complexed withpolyethylene glycol (PEG), metal ions, or incorporated into polymericcompounds such as polyacetic acid, polyglycolic acid, hydrogels,dextran, etc., or incorporated into liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of TRAIL-R, and are thus chosen according to the intendedapplication. TRAIL-R expressed on the surface of a cell may find use, aswell.

Compositions of the present invention may contain a TRAIL-R polypeptidein any form described herein, such as native proteins, variants,derivatives, oligomers, and biologically active fragments. In particularembodiments, the composition comprises a soluble TRAIL-R polypeptide oran oligomer comprising soluble TRAIL-R polypeptides.

TRAIL-R can be administered in any suitable manner, e.g., topically,parenterally, or by inhalation. The term "parenteral" includesinjection, e.g., by subcutaneous, intravenous, or intramuscular routes,also including localized administration, e.g., at a site of disease orinjury. Sustained release from implants is also contemplated. Oneskilled in the pertinent art will recognize that suitable dosages willvary, depending upon such factors as the nature of the disorder to betreated, the patient's body weight, age, and general condition, and theroute of administration. Preliminary doses can be determined accordingto animal tests, and the scaling of dosages for human administration areperformed according to art-accepted practices.

Compositions comprising TRAIL-R nucleic acids in physiologicallyacceptable formulations are also contemplated. TRAIL-R DNA may beformulated for injection, for example.

Antibodies

Antibodies that are immunoreactive with TRAIL-R polypeptides areprovided herein. Such antibodies specifically bind TRAIL-R, in that theantibodies bind to TRAIL-R via the antigen-binding sites of the antibody(as opposed to non-specific binding).

The TRAIL-R protein prepared as described in example 1 may be employedas an immunogen in producing antibodies immunoreactive therewith.Alternatively, another form of TRAIL-R, such as a fragment or fusionprotein, is employed as the immunogen.

Polyclonal and monoclonal antibodies may be prepared by conventionaltechniques. See, for example, Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Kennet et al. (eds.), Plenum Press,New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land(eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1988). Production of monoclonal antibodies directed against TRAIL-R isfurther illustrated in example 4.

Antigen-binding fragments of such antibodies, which may be produced byconventional techniques, are also encompassed by the present invention.Examples of such fragments include, but are not limited to, Fab andF(ab')₂ fragments. Antibody fragments and derivatives produced bygenetic engineering techniques are also provided.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies may be prepared by known techniques, and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993).

Among the uses of the antibodies is use in assays to detect the presenceof TRAIL-R polypeptides, either in vitro or in vivo. The antibodies alsomay be employed in purifying TRAIL-R proteins by immunoaffinitychromatography.

Those antibodies that additionally can block binding of TRAIL-R to TRAILmay be used to inhibit a biological activity that results from suchbinding. Such blocking antibodies may be identified using any suitableassay procedure, such as by testing antibodies for the ability toinhibit binding of TRAIL to cells expressing TRAIL-R. Examples of suchcells are the Jurkat cells and PSI cells described in example 2 below.Alternatively, blocking antibodies may be identified in assays for theability to inhibit a biological effect that results from binding ofTRAIL to target cells. Antibodies may be assayed for the ability toinhibit TRAIL-mediated lysis of Jurkat cells, for example.

Such an antibody may be employed in an in vitro procedure, oradministered in vivo to inhibit a TRAIL-R-mediated biological activity.Disorders caused or exacerbated (directly or indirectly) by theinteraction of TRAIL with cell surface TRAIL receptor thus may betreated. A therapeutic method involves in vivo administration of ablocking antibody to a mammal in an amount effective in inhibiting aTRAIL-mediated biological activity. Disorders caused or exacerbated byTRAIL, directly or indirectly, are thus treated. Monoclonal antibodiesare generally preferred for use in such therapeutic methods. In oneembodiment, an antigen-binding antibody fragment is employed.

A blocking antibody directed against TRAIL-R may be substituted forTRAIL-R in the above-described method of treating thromboticmicroangiopathy, e.g., in treating TTP or HUS. The antibody isadministered in vivo, to inhibit TRAIL-mediated damage to (e.g.,apoptosis of) microvascular endothelial cells.

Antibodies raised against TRAIL-R may be screened for agonistic (i.e.,ligand-mimicking) properties. Such antibodies, upon binding to cellsurface TRAIL-R, induce biological effects (e.g., transduction ofbiological signals) similar to the biological effects induced when TRAILbinds to cell surface TRAIL-R. Agonistic antibodies may be used toinduce apoptosis of certain cancer cells or virally infected cells, ashas been reported for TRAIL. The ability of TRAIL to kill cancer cells(including but not limited to leukemia, lymphoma, and melanoma cells)and virally infected cells is described in Wiley et al. (Immunity3:673-682, 1995); and in PCT application WO 97/01633.

Compositions comprising an antibody that is directed against TRAIL-R,and a physiologically acceptable diluent, excipient, or carrier, areprovided herein. Suitable components of such compositions are asdescribed above for compositions containing TRAIL-R proteins.

Also provided herein are conjugates comprising a detectable (e.g.,diagnostic) or therapeutic agent, attached to an antibody directedagainst TRAIL-R. Examples of such agents are presented above. Theconjugates find use in in vitro or in vivo procedures.

Nucleic Acids

The present invention provides TRAIL-R nucleic acids. Such nucleic acidsinclude, but are not limited to, DNA encoding the peptide described inexample 2. Such DNAs can be identified from knowledge of the geneticcode. Other nucleic acids of the present invention include isolated DNAscomprising the nucleotide sequence presented in SEQ ID NO:1 or SEQ IDNO:3.

The present invention provides isolated nucleic acids useful in theproduction of TRAIL-R polypeptides, e.g., in the recombinant expressionsystems discussed above. Such nucleic acids include, but are not limitedto, the human TRAIL-R DNA of SEQ ID NO:1. Nucleic acid molecules of thepresent invention include TRAIL-R DNA in both single-stranded anddouble-stranded form, as well as the RNA complement thereof. TRAIL-R DNAincludes, for example, cDNA, genomic DNA, chemically synthesized DNA,DNA amplified by PCR, and combinations thereof. Genomic DNA may beisolated by conventional techniques, e.g., using the cDNA of SEQ ID NO:1or 3, or a suitable fragment thereof, as a probe.

DNAs encoding TRAIL-R in any of the forms contemplated herein (e.g.,full length TRAIL-R or fragments thereof) arc provided. Particularembodiments of TRAIL-R-encoding DNAs include a DNA encoding the fulllength human TRAIL-R of SEQ ID NO:2 (including the N-terminal signalpeptide), and a DNA encoding a full length mature human TRAIL-R. Otherembodiments include DNA encoding a soluble TRAIL-R (e.g., encoding theextracellular domain of the protein of SEQ ID NO:2, either with orwithout the signal peptide).

One embodiment of the invention is directed to fragments of TRAIL-Rnucleotide sequences comprising at least about 17 contiguous nucleotidesof a TRAIL-R DNA sequence. In other embodiments, a DNA fragmentcomprises at least 30, or at least 60, contiguous nucleotides of aTRAIL-R DNA sequence. Nucleic acids provided herein include DNA and RNAcomplements of said fragments, along with both single-stranded anddouble-stranded forms of the TRAIL-R DNA.

Among the uses of TRAIL-R nucleic acid fragments is use as probes orprimers. Using knowledge of the genetic code in combination with theamino acid sequences set forth in example 2, sets of degenerateoligonucleotides can be prepared. Such oligonucleotides find use asprimers, e.g., in polymerase chain reactions (PCR), whereby TRAIL-R DNAfragments arc isolated and amplified.

Other useful fragments of the TRAIL-R nucleic acids include antisense orsense oligonucleotides comprising a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target TRAIL-R mRNA(sense) or TRAIL-R DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of TRAIL-R DNA. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 toabout 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of TRAIL-Rproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10448, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄ -mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

The following examples are provided to further illustrate particularembodiments of the invention, and are not to be construed as limitingthe scope of the present invention.

EXAMPLE 1 Purification of TRAIL-R Protein

A human TRAIL receptor (TRAIL-R) protein was prepared by the followingprocedure. Trail-R was isolated from the cell membranes of Jurkat cells,a human acute T leukemia cell line. Jurkat cells were chosen because aspecific band can be affinity precipitated from surface-biotinylatedJurkat cells, using Flag® TRAIL covalently coupled to affi-gel beads(Biorad Laboratories, Richmond, Calif.). The precipitated band has amolecular weight of about 52 kD. A minor specific band of about 42 kDalso was present, possibly accounting for a proteolytic breakdownproduct or a less glycosylated form of TRAIL-R.

Approximately 50 billion Jurkat cells were harvested by centrifugation(80 ml of cell pellet), washed once with PBS, then shock frozen onliquid nitrogen. Plasma membranes were isolated according to methodnumber three described in Maeda et al. Biochim. el Biophys. Acta,731:115, 1983; hereby incorporated by reference) with fivemodifications:

1. The following protease inhibitors were included in all solutions atthe indicated concentrations: Aprotinin, 150 nM; EDTA, 5 mM; Leupeptin,1 μM; pA-PMSF, 20 μM; Pefabloc, 500 μM; Pepstatin A, 1 μM; PMSF, 500 μM.

2. Dithiothreitol was not used.

3. DNAase was not used in the homogenization solution.

4. 1.25 ml of homogenization buffer was used per ml of cell pellet.

5. The homogenization was accomplished by five passages through a groundglass dounce homogenizer.

After isolation of the cell membranes, proteins were solubilized byresuspending the isolated membranes in 50 ml PBS containing 1%octylglucoside and all of the above mentioned protease inhibitors at theabove indicated concentrations. The resulting solution was thenrepeatedly vortexed during a thirty-minute incubation at 4° C. Thesolution was then centrifuged at 20,000 rpm in an SW28 rotor in an LE-80Beckman ultracentrifuge (Beckman Instruments, Inc., Palo Alto, Calif.)at 4° C. for 30 minutes to obtain the supernatant (the membraneextract).

Chromatography

The first step of purification of TRAIL-R out of the membrane extractprepared above was affinity chromatography. The membrane extract wasfirst applied to an anti-Flag® M2 affi-gel column (10 mg of monoclonalantibody M2 coupled to 2 ml of Affi-gel beads) to adsorb anynonspecifically binding material. The flow-through was then applied to aFlag®-TRAIL affi-gel column (10 mg of recombinant protein coupled to 1ml of affi-gel beads).

The Affi-gel support is an N-hydroxysuccinimide ester of a derivatized,crosslinked agarose gel bead (available from Biorad Laboratories,Richmond, Calif.). As discussed above, the Flag® peptide,Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, provides an epitope reversibly bound byspecific monoclonal antibodies, enabling rapid assay and facilepurification of expressed recombinant protein. M2 is a monoclonalantibody that binds Flag®. Monoclonal antibodies that bind the Flag®peptide, as well as other reagents for preparing and using Flag® fusionproteins, are available from Eastman Kodak Colo., Scientific ImagingSystems Division, New Haven, Conn. Preparation of Flag®-TRAIL fusionproteins (comprising Flag® fused to a soluble TRAIL polypeptide) isfurther described in PCT application WO 97/01633, hereby incorporated byreference.

The column was washed with 25 ml of each of the following buffers, inthe order indicated:

1. PBS containing 1% octylglucoside

2. PBS

3. PBS containing an additional 200 mM NaCl

4. PBS

The bound material was eluted with 50 mM Na Citrate (pH 3) in 1 mlfractions and immediately neutralized with 300 μl of 1 M Tris-HCl (pH8.5) per fraction. The TRAIL-binding activity of each fraction wasdetermined by a TRAIL-R-specific ELISA as described below. Fractionswith high TRAIL-binding activity were pooled, brought to 0.1%Trifluoroacetic acid (TFA), and subsequently chromatographed on acapillary reversed-phase HPLC column [500 μm internal diameter×25 cmfused silicone capillary column packed with 5 μm Vydac C₄ material(Vydac, Hesperia, Calif.)] using a linear gradient (2% per minute) from0% to 100% acetonitrile in water containing 0.1% TFA. Fractionscontaining high TRAIL-binding activity are then determined as above,pooled, and, if desired, lyophilized.

TRAIL-R-specific ELISA:

Serial dilutions of TRAIL-R-containing samples (in 50 mM NaHCO₃, broughtto pH 9 with NaOH) were coated onto Linbro/Titertek 96 well flat bottomE.I.A. microtitration plates (ICN Biomedicals Inc., Aurora, Ohio) at 100μl/well. After incubation at 4° C. for 16 hours, the wells were washedsix times with 200 μl PBS containing 0.05% Tween-20 (PBS-Tween). Thewells were then incubated with Flag®-TRAIL at 1 μg/ml in PBS-Tween with5% fetal calf serum (FCS) for 90 minutes (100 μl per well), followed bywashing as above. Next, each well was incubated with the anti-Flag®,monoclonal antibody M2 at 1 μg/ml in PBS-Tween containing 5% FCS for 90minutes (100 μl per well), followed by washing as above. Subsequently,wells were incubated with a polyclonal goat anti-mIgGI-specifichorseradish peroxidase-conjugated antibody (a 1:5000 dilution of thecommercial stock in PBS-Tween containing 5% FCS) for 90 minutes (100 μlper well). The HRP-conjugated antibody was obtained from SouthernBiotechnology Associates, Inc., Birmingham, Ala. Wells then were washedsix times, as above.

For development of the ELISA, a substrate mix [100 μl per well of a 1:1premix of the TMB Peroxidase Substrate and Peroxidase Solution B(Kirkegaard Perry Laboratories, Gaithersburg, Md.)] was added to thewells. After sufficient color reaction, the enzymatic reaction wasterminated by addition of 2 N H₂ SO₄ (50 μl per well). Color intensity(indicating TRAIL-binding activity) was determined by measuringextinction at 450 nm on a V Max plate reader (Molecular Devices,Sunnyvale, Calif.).

EXAMPLE 2 Amino Acid Sequence

(a) TRAIL-R purified from Jurkat cells

TRAIL-R protein isolated from Jurkat cells was digested with trypsin,using conventional procedures. Amino acid sequence analysis wasconducted on one of the peptide fragments produced by the trypticdigest. The fragment was found to contain the following sequence, whichcorresponds to amino acids 327 to 333 of the sequence presented in SEQID NO:2: VPANEGD.

(b) TRAIL-R purified from PS-1 cells

TRAIL-R protein was also isolated from PS-1 cells. PS-1 is a human Bcell line that spontaneously arose after lethal irradiation of humanperipheral blood lymphocytes (PBLs). The TRAIL-R protein was digestedwith trypsin, using conventional procedures. Amino acid sequenceanalysis was conducted on peptide fragments that resulted from thetryptic digest. One of the fragments was found to contain the followingsequence, which, like the fragment presented in (a), corresponds toamino acids 327 to 333 of the sequence presented in SEQ ID NO:2:VPANEGD.

EXAMPLE 3 DNA and Amino Acid Sequences

The amino acid sequence of additional tryptic digest peptide fragmentsof TRAIL-R was determined. Degenerate oligonucleotides, based upon theamino acid sequence of two of the peptides, were prepared. A TRAIL-R DNAfragment was isolated and amplified by polymerase chain reaction (PCR),using the degenerate oligonucleotides as 5' and 3' primers. The PCR wasconducted according to conventional procedures, using cDNA derived fromthe PS-1 cell line described in example 2 as the template. Thenucleotide sequence of the isolated TRAIL-R DNA fragment (excludingportions corresponding to part of the primers), and the amino acidsequence encoded thereby, are presented in FIG. 1 (SEQ ID NOS:3 and 4).The sequence of the entire TRAIL-R DNA fragment isolated by PCRcorresponds to nucleotides 988 to 1164 of SEQ ID NO:1, which encodeamino acids 330 to 388 of SEQ ID NO:2.

The amino acid sequence in SEQ ID NO:4 bears significant homology to theso-called death domains found in certain other receptors. Thecytoplasmic region of Fas and TNF receptor type I each contain a deathdomain, which is associated with transduction of an apoptotic signal(Tartaglia et al. Cell 74:845, 1993; Itoh and Nagata, J. Biol. Chem.268:10932, 1993). Thus, the sequence presented in SEQ ID NO:4 isbelieved to be found within the cytoplasmic domain of TRAIL-R.

A probe derived from the fragment isolated above was used to screen acDNA library (human foreskin fibroblast-derived cDNA in λgt10 vector),and a human TRAIL-R cDNA was isolated. The nucleotide sequence of thecoding region of this cDNA is presented in SEQ ID NO: 1, and the aminoacid sequence encoded thereby is shown in SEQ ID NO:2.

EXAMPLE 4 Monoclonal Antibodies That Bind TRAIL-R

This example illustrates a method for preparing monoclonal antibodiesthat bind TRAIL-R. Suitable immunogens that may be employed ingenerating such antibodies include, but are not limited to, purifiedTRAIL-R protein or an immunogenic fragment thereof such as theextracellular domain, or fusion proteins containing TRAIL-R (e.g., asoluble TRAIL-R/Fc fusion protein).

Purified TRAIL-R can be used to generate monoclonal antibodiesimmunoreactive therewith, using conventional techniques such as thosedescribed in U.S. Pat. No. 4,411,993. Briefly, mice are immunized withTRAIL-R immunogen emulsified in complete Freund's adjuvant, and injectedin amounts ranging from 10-100 μg subcutaneously or intraperitoneally.Ten to twelve days later, the immunized animals are boosted withadditional TRAIL-R emulsified in incomplete Freund's adjuvant. Mice areperiodically boosted thereafter on a weekly to bi-weekly immunizationschedule. Serum samples are periodically taken by retro-orbital bleedingor tail-tip excision to test for TRAIL-R antibodies by dot blot assay,ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of TRAILbinding.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of TRAIL-R in saline. Threeto four days later, the animals are sacrificed, spleen cells harvested,and spleen cells are fused to a murine myeloma cell line, e.g., NS1 orpreferably P3x63Ag8.653 (ATCC CRL 1580). Fusions generate hybridomacells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified TRAIL-R by adaptations of the techniques disclosed in Engvallet al., Immunochem. 8:871, 1971 and in U.S. Pat. No. 4,703,004. Apreferred screening technique is the antibody capture techniquedescribed in Beckmann et al., (J. Immunol. 144:4212, 1990) Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-TRAIL-Rmonoclonal antibodies. Alternatively, hybridoma cells can be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to Protein A orProtein G can also be used, as can affinity chromatography based uponbinding to TRAIL-R.

EXAMPLE 5 Northern Blot Analysis

The tissue distribution of TRAIL-R mRNA was investigated by Northernblot analysis, as follows. An aliquot of a radiolabeled probe (the sameradiolabeled probe used to screen the cDNA library in example 3) wasadded to two different human multiple tissue Northern blots (Clontech,Palo Alto, Calif.; Biochain, Palo Alto, Calif.). Hybridization wasconducted overnight at 63° C. in 50% formamide as previously described(March et al., Nature 315:641-647, 1985). The blots then were washedwith 2× SSC, 0.1% SDS at 68° C. for 30 minutes. Aglycerol-aldehyde-phosphate dehydrogenase (GAPDH) specific probe wasused to standardize for RNA loadings.

A single transcript of 4.4 kilobases (kb) was present in all tissuesexamined, including spleen, thymus, peripheral blood lymphocytes (PBIs),prostate, testis, ovary, uterus, placenta, and multiple tissues alongthe gastro-intestinal tract (including esophagus, stomach, duodenum,jejunum/ileum, colon, rectum, and small intestine). The cells andtissues with the highest levels of TRAIL-R mRNA are PBLs, spleen, andovary, as shown by comparison to control hybridizations with aGAPDH-specific probe.

EXAMPLE 6 Binding Assay

Full length human TRAIL-R was expressed and tested for the ability tobind TRAIL. The binding assay was conducted as follows.

A fusion protein comprising a leucine zipper peptide fused to theN-terminus of a soluble TRAIL polypeptide (LZ-TRAIL) was employed in theassay. An expression construct was prepared, essentially as describedfor preparation of the Flag®-TRAIL expression construct in Wiley et al.(Immunity, 3:673-682, 1995; hereby incorporated by reference), exceptthat DNA encoding the Flag® peptide was replaced with a sequenceencoding a modified leucine zipper that allows for trimerization. Theconstruct, in expression vector pDC409, encoded a leader sequencederived from human cytomegalovirus, followed by the leucine zippermoiety fused to the N-terminus of a soluble TRAIL polypeptide. The TRAILpolypeptide comprised amino acids 95-281 of human TRAIL (a fragment ofthe extracellular domain), as described in Wiley et al. (supra). TheLZ-TRAIL was expressed in CHO cells, and purified from the culturesupernatant.

The expression vector designated pDC409 is a mammalian expression vectorderived from the pDC406 vector described in McMahan et al. (EMBO J.10:2821-2832, 1991; hereby incorporated by reference). Features added topDC409 (compared to pDC406) include additional unique restriction sitesin the multiple cloning site (mcs); three stop codons (one in eachreading frame) positioned downstream of the mcs; and a T7 polymerasepromoter, downstream of the mcs, that faciliates sequencing of DNAinserted into the mcs.

For expression of full length human TRAIL-R protein, the entire codingregion (i.e., the DNA sequence presented in SEQ ID NO:1) was amplifiedby polymerase chain reaction (PCR). The template employed in the PCR wasthe cDNA clone isolated from a human foreskin fibroblast cDNA library,as described in example 3. The isolated and amplified DNA was insertedinto the expression vector pDC409, to yield a construct designatedpDC409-TRAIL-R.

CrmA protein was employed to inhibit apoptosis of host cells expressingrecombinant TRAIL-R, as discussed above and in example 8. An expressionvector designated pDC409-CrmA contained DNA encoding poxvirus CrmA inpDC409. The 38-kilodalton cowpox-derived protein that was subsequentlydesignated CrmA is described in Pickup et al. (Proc. Natl. Acad. Sci.USA 83:7698-7702, 1986; hereby incorporated by reference).

CV-1/EBNA cells were co-transfected with pDC409-TRAIL-R together withpDC409-CrmA, or with pDC409-CrmA alone. The cells were cultured in DMEMsupplemented with 10% fetal bovine serum, penicillin, streptomycin, andglutamine. 48 hours after transfection, cells were detachednon-enzymatically and incubated with LZ-TRAIL (5 μg/ml), a biotinylatedanti-LZ monoclonal antibody (5 μg/ml), and phycoerythrin-conjugatedstreptavidin (1:400), before analysis by fluorescence-activated cellscanning (FACS). The cytometric analysis was conducted on a FACscan(Beckton Dickinson, San Jose, Calif.).

The CV-1/EBNA cells co-transfected with vectors encoding TRAIL-R andCrmA showed significantly enhanced binding of LZ-TRAIL, compared to thecells transfected with the CrmA-encoding vector alone.

EXAMPLE 7 TRAIL-R Blocks TRAIL-Induced Apoptosis of Target Cells

TRAIL-R was tested for the ability to block TRAIL-induced apoptosis ofJurkat cells. The TRAIL-R employed in the assay was in the form of afusion protein designated sTRAIL-R/Fc, which comprised the extracellulardomain of human TRAIL-R, fused to the N-terminus of an Fc polypeptidederived from human IgG1.

CV1-EBNA cells were transfected with a recombinant expression vectorcomprising a gene fusion encoding the sTRAIL-R/Fc protein, in the pDC409vector described in example 6, and cultured to allow expression of thefusion protein. The sTRAIL-R/Fc fusion protein was recovered from theculture supernatant. Procedures for fusing DNA encoding an IgG1 Fepolypeptide to DNA encoding a heterologous protein are described inSmith et al., (Cell 73:1349-1360, 1993); analogous procedures wereemployed herein.

A fusion protein designated TNF-R2-Fc, employed as a control, comprisedthe extracellular domain of the TNF receptor protein known as p75 or p80TNF-R (Smith et al., Science 248:1019-1023, 1990; Smith et al. Cell76:959-962, 1994), fused to an Fc polypeptide. A mouse monoclonalantibody that is a blocking antibody directed against human TRAIL, wasemployed in the assay as well.

Jurkat cells were incubated with varying or constant concentrations ofLZ-TRAIL (the LZ-TRAIL fusion protein described in example 6), in theabsence or presence of varying concentrations of sTRAIL-R-Fc, TNF-R2-Fc,or the TRAIL-specific monoclonal antibody. Cell death was quantitatedusing an MTT cell viability assay (MTT is ³-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), aspreviously described (Mosmann, J. Immunol. Methods 65:55-63, 1983). Theresults are shown in FIG. 2, which presents the percent cell death forJurkat cells that were untreated (A) or were treated with varying (▴) orconstant (∘, , □, ▪) concentrations of LZ-TRAIL (13 ng/ml) in theabsence () or presence of varying concentrations of TRAIL-R2-Fc (▪),TNF-R2-Fc (□), or the anti-TRAIL antibody (∘). Varying concentrationsfor all substances started at 10 μg/ml and were serially diluted.

The anti-TRAIL monoclonal antibody and sTRAIL-R/Fc each blockedTRAIL-induced apoptosis in a dose dependent fashion, whereas TNFR2-Fcdid not. Thus, the extracellular domain of TRAIL-R is capable of bindingto TRAIL and inhibiting TRAIL-mediated apoptosis of target cells.

EXAMPLE 8 TRAIL-R-Induced Apoptosis is Blocked by Caspase Inhibitors andFADD-DN

CV-1/EBNA cells were transfected, by the DEAE-dextran method, withexpression plasmids for TRAIL-R (pDC409-TRAIL-R), together with athreefold excess of empty expression vector (pDC409) in the presence orabsence of z-VAD-fmk (10 μM; in the culture medium), or together with athreefold excess of expression vector pDC409-CrmA, pDC409-p35, orpDC409-FADD-DN. In addition, 400 ng/slide of an expression vector forthe E. coli lacz gene was co-transfected together with all DNA mixes.The transfected cells were cultured in chambers mounted on slides.

The mammalian expression vector pDC409, and the pDC409-TRAIL-R vectorencoding full length human TRAIL-R, are described in example 6. Thetripeptide derivative zVAD-fmk(benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone) is available fromEnzyme System Products, Dublin, Calif.

The 38-kilodalton cowpox-derived protein that was subsequentlydesignated CrmA is described in Pickup et al. (Proc. Natl. Acad. Sci.USA 83:7698-7702, 1986; hereby incorporated by reference). Sequenceinformation for CrmA is presented in FIG. 4 of Pickup et al., supra.

A 35-kilodalton protein encoded by Autographa californica nuclearpolyhedrosis virus, a baculovirus, is described in Friesen and Miller(J. Virol. 61:2264-2272, 1987; hereby incorporated by reference).Sequence information for this protein, designated baculovirus p35herein, is presented in FIG. 5 of Friesen and Miller, supra.

FADD (also designated MORT1) is described in Boldin et al. (J. Biol.Chem. 270:7795-7798, 1995; hereby incorporated by reference). Theprotein referred to as FADD-DN (FADD dominant negative) is a C-terminalfragment of FADD that includes the death domain. DNA encoding FADD-DN,fused to an N-terminal Flag® epitope tag (described above), was insertedinto the pDC409 expression vector described in example 6, to formpDC409-FADD-DN. The FADD-DN polypeptide corresponds to amino acids 117through 245 of the MORT1 amino acid sequence presented in Boldin et al.,supra.

48 hours after transfection, cells were washed with PBS, fixed withglutaraldehyde and incubated with X-gal(5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside). Cells expressingβ-galactosidase stain blue. A decrease in the percentage of stainedcells indicates loss of β-galactosidase expression, and correlates withdeath of cells that express the protein(s) co-transfected with the laczgene.

The results are presented in FIG. 3, wherein the values plottedrepresent the mean and standard deviation of at least three separateexperiments. Poxvirus CrmA, baculovirus p35, FADD-DN, and z-VAD-fmk eacheffectively reduced death of transfected cells expressing TRAIL-R.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 5                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1323 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: huTrail-R                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1323                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATG GAA CAA CGG GGA CAG AAC GCC CCG GCC GC - #T TCG GGG GCC CGG AAA           48                                                                          Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Al - #a Ser Gly Ala Arg Lys           #                 15                                                          - AGG CAC GGC CCA GGA CCC AGG GAG GCG CGG GG - #A GCC AGG CCT GGG CCC           96                                                                          Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gl - #y Ala Arg Pro Gly Pro           #             30                                                              - CGG GTC CCC AAG ACC CTT GTG CTC GTT GTC GC - #C GCG GTC CTG CTG TTG          144                                                                          Arg Val Pro Lys Thr Leu Val Leu Val Val Al - #a Ala Val Leu Leu Leu           #         45                                                                  - GTC TCA GCT GAG TCT GCT CTG ATC ACC CAA CA - #A GAC CTA GCT CCC CAG          192                                                                          Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gl - #n Asp Leu Ala Pro Gln           #     60                                                                      - CAG AGA GCG GCC CCA CAA CAA AAG AGG TCC AG - #C CCC TCA GAG GGA TTG          240                                                                          Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Se - #r Pro Ser Glu Gly Leu           # 80                                                                          - TGT CCA CCT GGA CAC CAT ATC TCA GAA GAC GG - #T AGA GAT TGC ATC TCC          288                                                                          Cys Pro Pro Gly His His Ile Ser Glu Asp Gl - #y Arg Asp Cys Ile Ser           #                 95                                                          - TGC AAA TAT GGA CAG GAC TAT AGC ACT CAC TG - #G AAT GAC CTC CTT TTC          336                                                                          Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Tr - #p Asn Asp Leu Leu Phe           #           110                                                               - TGC TTG CGC TGC ACC AGG TGT GAT TCA GGT GA - #A GTG GAG CTA AGT CCG          384                                                                          Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Gl - #u Val Glu Leu Ser Pro           #       125                                                                   - TGC ACC ACG ACC AGA AAC ACA GTG TGT CAG TG - #C GAA GAA GGC ACC TTC          432                                                                          Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cy - #s Glu Glu Gly Thr Phe           #   140                                                                       - CGG GAA GAA GAT TCT CCT GAG ATG TGC CGG AA - #G TGC CGC ACA GGG TGT          480                                                                          Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Ly - #s Cys Arg Thr Gly Cys           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - CCC AGA GGG ATG GTC AAG GTC GGT GAT TGT AC - #A CCC TGG AGT GAC ATC          528                                                                          Pro Arg Gly Met Val Lys Val Gly Asp Cys Th - #r Pro Trp Ser Asp Ile           #               175                                                           - GAA TGT GTC CAC AAA GAA TCA GGT ACA AAG CA - #C AGT GGG GAA GCC CCA          576                                                                          Glu Cys Val His Lys Glu Ser Gly Thr Lys Hi - #s Ser Gly Glu Ala Pro           #           190                                                               - GCT GTG GAG GAG ACG GTG ACC TCC AGC CCA GG - #G ACT CCT GCC TCT CCC          624                                                                          Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gl - #y Thr Pro Ala Ser Pro           #       205                                                                   - TGT TCT CTC TCA GGC ATC ATC ATA GGA GTC AC - #A GTT GCA GCC GTA GTC          672                                                                          Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Th - #r Val Ala Ala Val Val           #   220                                                                       - TTG ATT GTG GCT GTG TTT GTT TGC AAG TCT TT - #A CTG TGG AAG AAA GTC          720                                                                          Leu Ile Val Ala Val Phe Val Cys Lys Ser Le - #u Leu Trp Lys Lys Val           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - CTT CCT TAC CTG AAA GGC ATC TGC TCA GGT GG - #T GGT GGG GAC CCT GAG          768                                                                          Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gl - #y Gly Gly Asp Pro Glu           #               255                                                           - CGT GTG GAC AGA AGC TCA CAA CGA CCT GGG GC - #T GAG GAC AAT GTC CTC          816                                                                          Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Al - #a Glu Asp Asn Val Leu           #           270                                                               - AAT GAG ATC GTG AGT ATC TTG CAG CCC ACC CA - #G GTC CCT GAG CAG GAA          864                                                                          Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gl - #n Val Pro Glu Gln Glu           #       285                                                                   - ATG GAA GTC CAG GAG CCA GCA GAG CCA ACA GG - #T GTC AAC ATG TTG TCC          912                                                                          Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gl - #y Val Asn Met Leu Ser           #   300                                                                       - CCC GGG GAG TCA GAG CAT CTG CTG GAA CCG GC - #A GAA GCT GAA AGG TCT          960                                                                          Pro Gly Glu Ser Glu His Leu Leu Glu Pro Al - #a Glu Ala Glu Arg Ser           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - CAG AGG AGG AGG CTG CTG GTT CCA GCA AAT GA - #A GGT GAT CCC ACT GAG         1008                                                                          Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Gl - #u Gly Asp Pro Thr Glu           #               335                                                           - ACT CTG AGA CAG TGC TTC GAT GAC TTT GCA GA - #C TTG GTG CCC TTT GAC         1056                                                                          Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala As - #p Leu Val Pro Phe Asp           #           350                                                               - TCC TGG GAG CCG CTC ATG AGG AAG TTG GGC CT - #C ATG GAC AAT GAG ATA         1104                                                                          Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Le - #u Met Asp Asn Glu Ile           #       365                                                                   - AAG GTG GCT AAA GCT GAG GCA GCG GGC CAC AG - #G GAC ACC TTG TAC ACG         1152                                                                          Lys Val Ala Lys Ala Glu Ala Ala Gly His Ar - #g Asp Thr Leu Tyr Thr           #   380                                                                       - ATG CTG ATA AAG TGG GTC AAC AAA ACC GGG CG - #A GAT GCC TCT GTC CAC         1200                                                                          Met Leu Ile Lys Trp Val Asn Lys Thr Gly Ar - #g Asp Ala Ser Val His           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - ACC CTG CTG GAT GCC TTG GAG ACG CTG GGA GA - #G AGA CTT GCC AAG CAG         1248                                                                          Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Gl - #u Arg Leu Ala Lys Gln           #               415                                                           - AAG ATT GAG GAC CAC TTG TTG AGC TCT GGA AA - #G TTC ATG TAT CTA GAA         1296                                                                          Lys Ile Glu Asp His Leu Leu Ser Ser Gly Ly - #s Phe Met Tyr Leu Glu           #           430                                                               #           1323   CT GCC ATG TCC TAA                                         Gly Asn Ala Asp Ser Ala Met Ser  *                                            #       440                                                                   - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 440 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Al - #a Ser Gly Ala Arg Lys         #                 15                                                          - Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gl - #y Ala Arg Pro Gly Pro         #             30                                                              - Arg Val Pro Lys Thr Leu Val Leu Val Val Al - #a Ala Val Leu Leu Leu         #         45                                                                  - Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gl - #n Asp Leu Ala Pro Gln         #     60                                                                      - Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Se - #r Pro Ser Glu Gly Leu         # 80                                                                          - Cys Pro Pro Gly His His Ile Ser Glu Asp Gl - #y Arg Asp Cys Ile Ser         #                 95                                                          - Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Tr - #p Asn Asp Leu Leu Phe         #           110                                                               - Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Gl - #u Val Glu Leu Ser Pro         #       125                                                                   - Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cy - #s Glu Glu Gly Thr Phe         #   140                                                                       - Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Ly - #s Cys Arg Thr Gly Cys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Pro Arg Gly Met Val Lys Val Gly Asp Cys Th - #r Pro Trp Ser Asp Ile         #               175                                                           - Glu Cys Val His Lys Glu Ser Gly Thr Lys Hi - #s Ser Gly Glu Ala Pro         #           190                                                               - Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gl - #y Thr Pro Ala Ser Pro         #       205                                                                   - Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Th - #r Val Ala Ala Val Val         #   220                                                                       - Leu Ile Val Ala Val Phe Val Cys Lys Ser Le - #u Leu Trp Lys Lys Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gl - #y Gly Gly Asp Pro Glu         #               255                                                           - Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Al - #a Glu Asp Asn Val Leu         #           270                                                               - Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gl - #n Val Pro Glu Gln Glu         #       285                                                                   - Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gl - #y Val Asn Met Leu Ser         #   300                                                                       - Pro Gly Glu Ser Glu His Leu Leu Glu Pro Al - #a Glu Ala Glu Arg Ser         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Gl - #u Gly Asp Pro Thr Glu         #               335                                                           - Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala As - #p Leu Val Pro Phe Asp         #           350                                                               - Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Le - #u Met Asp Asn Glu Ile         #       365                                                                   - Lys Val Ala Lys Ala Glu Ala Ala Gly His Ar - #g Asp Thr Leu Tyr Thr         #   380                                                                       - Met Leu Ile Lys Trp Val Asn Lys Thr Gly Ar - #g Asp Ala Ser Val His         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Gl - #u Arg Leu Ala Lys Gln         #               415                                                           - Lys Ile Glu Asp His Leu Leu Ser Ser Gly Ly - #s Phe Met Tyr Leu Glu         #           430                                                               - Gly Asn Ala Asp Ser Ala Met Ser                                             #       440                                                                   - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 157 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: internal                                            -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: huTrail-R fr - #ag                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..155                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - CT GAG ACT CTG AGA CAG TGC TTC GAT GAC TTT - # GCA GAC TTG GTG CCC            47                                                                          #Phe Ala Asp Leu Val Proys Phe Asp Asp                                        #  15                                                                         - TTT GAC TCC TGG GAG CCG CTC ATG AGG AAG TT - #G GGC CTC ATG GAC AAT           95                                                                          Phe Asp Ser Trp Glu Pro Leu Met Arg Lys Le - #u Gly Leu Met Asp Asn           #                 30                                                          - GAG ATA AAG GTG GCT AAA GCT GAG GCA GCG GG - #C CAC AGG GAC ACC TTG          143                                                                          Glu Ile Lys Val Ala Lys Ala Glu Ala Ala Gl - #y His Arg Asp Thr Leu           #             45                                                              #    157           T                                                          Xaa Thr Met Leu                                                                        50                                                                   - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 51 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Al - #a Asp Leu Val Pro Phe         #                 15                                                          - Asp Ser Trp Glu Pro Leu Met Arg Lys Leu Gl - #y Leu Met Asp Asn Glu         #             30                                                              - Ile Lys Val Ala Lys Ala Glu Ala Ala Gly Hi - #s Arg Asp Thr Leu Xaa         #         45                                                                  - Thr Met Leu                                                                      50                                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: FLAG peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Asp Tyr Lys Asp Asp Asp Asp Lys                                               1               5                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated TRAIL-R DNA, wherein said DNAcomprises the nucleotide sequence presented in SEQ ID NO:1.
 2. Anisolated TRAIL-R DNA, wherein said DNA encodes a polypeptide comprisingamino acids 1 to 440 of SEQ ID NO:2.
 3. An isolated TRAIL-R DNA thatencodes a polypeptide capable of binding TRAIL, wherein said polypeptidecomprises amino acids x to 440 of SEQ ID NO:2, wherein x represents aninteger from 51 through
 59. 4. A TRAIL-R DNA of claim 3, wherein saidpolypeptide comprises amino acids 54 to 440 of SEQ ID NO:2.
 5. Anisolated TRAIL-R DNA, wherein said DNA encodes a soluble TRAIL-Rpolypeptide comprising amino acids x to 210 of SEQ ID NO:2, wherein xrepresents an integer from 51 through
 59. 6. An expression vectorcomprising a DNA according to claim
 1. 7. An expression vectorcomprising a DNA according to claim
 2. 8. An expression vectorcomprising a DNA according to claim
 3. 9. An expression vectorcomprising a DNA according to claim
 3. 10. An expression vectorcomprising a DNA according to claim
 4. 11. A host cell transformed withan expression vector of claim
 6. 12. A host cell transformed with anexpression vector of claim
 7. 13. A host cell transformed with anexpression vector of claim
 8. 14. A host cell transformed with anexpression vector of claim
 9. 15. A host cell transformed with anexpression vector of claim
 10. 16. An isolated TRAIL-R DNA that encodesa polypeptide comprising amino acids 1 to 210 of SEQ ID NO:2.
 17. Anisolated TRAIL-R DNA of claim 5, encoding a polypeptide comprising theamino acid sequence presented as residues 54 to 210 of SEQ ID NO:2. 18.An expression vector comprising a DNA of claim
 16. 19. An expressionvector comprising a DNA of claim
 17. 20. A host cell transformed with anexpression vector of claim
 18. 21. A host cell transformed with anexpression vector of claim
 19. 22. A process for preparing a TRAIL-Rpolypeptide, comprising culturing a host cell of claim 11 underconditions that promote expression of TRAIL-R, and recovering theTRAIL-R polypeptide.
 23. A process for preparing a TRAIL-R polypeptide,comprising culturing a host cell of claim 12 under conditions thatpromote expression of TRAIL-R, and recovering the TRAIL-R polypeptide.24. A process for preparing a TRAIL-R polypeptide, comprising culturinga host cell of claim 13 under conditions that promote expression ofTRAIL-R, and recovering the TRAIL-R polypeptide.
 25. A process forpreparing a TRAIL-R polypeptide, comprising culturing a host cell ofclaim 16 under conditions that promote expression of TRAIL-R, andrecovering the TRAIL-R polypeptide.
 26. A process for preparing aTRAIL-R polypeptide, comprising culturing a host cell of claim 15 underconditions that promote expression of TRAIL-R, and recovering theTRAIL-R polypeptide.
 27. A process for preparing a TRAIL-R polypeptide,comprising culturing a host cell of claim 20 under conditions thatpromote expression of TRAIL-R, and recovering the TRAIL-R polypeptide.28. A process for preparing a TRAIL-R polypeptide, comprising culturinga host cell of claim 21 under conditions that promote expression ofTRAIL-R, and recovering the TRAIL-R polypeptide.
 29. An isolated DNAencoding a TRAIL-R polypeptide comprising the extracellular domain ofthe TRAIL-R of SEQ ID NO:2, wherein the C-terminus of said extracellulardomain is amino acid 210 of SEQ ID NO:2, and comprising all or part ofthe cytoplasmic domain of the TRAIL-R of SEQ ID NO:2, wherein saidpolypeptide lacks a transmembrane region.
 30. An expression vectorcomprising a DNA according to claim
 29. 31. A host cell transformed withan expression vector of claim
 30. 32. A process for preparing a TRAIL-Rpolypeptide, comprising culturing a host cell of claim 30 underconditions that promote expression of TRAIL-R, and recovering theTRAIL-R polypeptide.